The goal of the proposed research is to understand in molecular detail the synthesis of a hydrophobic protein and the means by which it rapidly and asymmetrically inserts into the proper biological membrane. The major coat protein of the virus M13 is an integral protein of the host cell membrane at all stages of infection and will therefore be used to study the membrane assembly process. It is initially made in infected cells by soluble polysomes as a procoat with 23 additional N-terminal residues; we propose to purify this procoat. Both in vivo and (recently) in vitro, procoat has been found to assemble into the cytoplasmic membrane and be proteolytically converted to coat protein. We have solubilized the leader peptidase responsible for this cleavage and propose to purify and characterize it. We also propose to identify, isolate, and reconstitute the factor(s) responsible for membrane-specific assembly in our cell-free reaction.